Project Summary Polymerases with ultra-high processivity would be exceptionally useful in biotechnology because they will make it possible to monitor long-range correlated changes in the sequence, structure and post-synthetic modifications of DNA or RNA without losing information on linkage between sites. We have discovered an ultra-processive reverse-transcriptase (MarathonRT) during the course of our structural studies on group II intron encoded proteins, and have been optimizing it for use as a new tool in the enzyme arsenal of molecular biologists and genomicists, particularly in studies of long-range correlated mutations within viral genomes such as HIV. Specifically, we are interested in understanding the patterns by which correlated mutations shift in response to antiretroviral drug treatment, thereby providing insights into mechanisms of resistance and, potentially, providing clinically valuable information on changes in therapeutic drug combinations for infected patients. While our ongoing parent grant continues to develop MarathonRT as a tool enzyme for a variety of applications in RNA science, in this supplemental study we will focus specifically on the use of MarathonRT for discovery of regulatory structures, sequences and post-transcriptional modifications within the HIV genome. We will conduct chemical probing studies, analysis of correlated mutations and studies on changes in RT dynamical behavior, such as pausing. These investigations will be carried out in conjunction with adaptations to emerging sequencing platforms, such as Oxford Nanopore MinION sequencing, enabling a unified and high- throughput approach for the discovery of specific regulatory elements in viruses. The work will have direct clinical applications to our understanding and treatment of drug resistance in patients, and it will lay the groundwork for related studies in other clinically relevant viruses, such as HCV.